This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission. Glutamate receptor signaling is organized by the membrane-associated guanylate kinase (MAGuK) family of scaffold proteins. Scaffolds determine the outcome of signal transduction by controlling the location of neurotransmitter receptors and connecting them to downstream effectors. In particular, two homologous MAGuKs (PSD-95 and PSD-93) play opposing roles in synaptic plasticity yet the basis for their differential activity remains a mystery. A key factor in synaptic plasticity is post-translation modification (PTM) of the MAGuK proteins, which have been missing in most studies that have been published to date. This proposal investigates the posttranslational regulation of PSD-95 and PSD-93. Aim 1 will test the hypothesis that phosphorylation alters the structure and activity of MAGuKs. We propose that a difference in the number and location of PTM sites elicits activity differences. Aim 2 will test the hypothesis that palmitoylation, a lipid modification essential for synaptic targeting, alters MAGuK activity and coverts them into the filament observed at synapses. Studies to date focused on soluble proteins but palmitoylation is indispensable for activity. Aim 3 will test the hypothesis that phase transitions of MAGuKs regulate availability for receptor binding. SynGAP, an essential synaptic protein, induces to liquid phase separation of PSD-95. This can generate a dynamic ?membrane-less organelle?. We will investigate the functional effects of phase separation on receptor binding and structure. We possess the only working reconstitution of scaffold interactions in the post-synapse and provided the only kinetic description of MAGuK scaffolding activity. We will use our unique expertise to address how PTM changes the activity of MAGuKs. We have pioneered novel methods for structural refinement of proteins containing intrinsic disorder and generated the only structural model of a full-length scaffold protein to date. We will use these approaches to provide a mechanistic understanding of how MAGuK structure affects scaffolding. Our published work suggests that proteins residing together on PSD-95 have ?higher-order? interactions driven by the enforced proximity. Describing the molecular events in excitatory signaling is a fundamental challenge in neuroscience with direct relevance to brain development, memory and learning, and many neurological and neuropsychiatric disorders.